XenWiki - User contributions [en]

Protocols

2011-11-23T12:29:08Z

68.147.37.142:

*'''''Books for Xenopus Research and Protocols'''''
**'''Normal Table of Xenopus Laevis (Daudin)''', Nieuwkoop and Faber (Third edition), 1994 [http://www.xenbase.org/literature/book.do?method=display&bookId=1]
***a systematic and chronological description of Xenopus laevis development
**'''Early Development of Xenopus laevis: A Laboratory Manual''', Sive, Grainger, and Harland, 2000 [http://www.xenbase.org/literature/book.do?method=display&bookId=22]
***comprehensive collection of protocols for the study of early development in Xenopus embryos
**'''Xenopus Protocols: Cell Biology and Signal Transduction''', Liu (First edition), 2006 [http://www.xenbase.org/literature/book.do?method=display&bookId=206]
***step-by-step laboratory instructions, an introduction outlining the principles behind the technique, lists of the necessary equipment and reagents
**'''Xenopus laevis: Practical Uses in Cell and Molecular Biology, Volume 36''', Kay and Peng, 1991 [http://www.xenbase.org/literature/book.do?method=display&bookId=204]
***detailed description of a wide range of uses for Xenopus laevis oocytes and embryos in cell and molecular biology
**'''Transgenic Xenopus: Microinjection Methods and Developmental Neurobiology''', Seidman and Soreq, 1997 [http://www.xenbase.org/literature/book.do?method=display&bookId=203]
***referenced guide to the use of microinjected embryos studying the role and regulation of nervous system proteins during development
**'''Atlas of Xenopus Development''', Bernardini, Prati, Bonetti, and Scari, 1999 [http://www.xenbase.org/literature/book.do?method=display&bookId=202]
***scanning, transmission, and light microscopy images of Xenopus embryonic development
**'''The early development of Xenopus laevis. An atlas of the histology''', Hausen and Riebesell, 1991 [http://www.xenbase.org/literature/book.do?method=display&bookId=124]
***detailed histological sections of Xenopus embryonic development
**'''Color Atlas of Xenopus laevis Histology''', Wiechmann and Wirsig-Wiechmann, 2003 [http://www.xenbase.org/literature/book.do?method=display&bookId=82]
***central source on the microscopic anatomy of cells, tissues, and major organs of Xenopus laevis

*'''''Animal Husbandry/Animal Protocols'''''
**[[inducing egg laying via hCG injection (Vize lab)]]
**[[in vitro fertilization of Xenopus laevis (Conlon lab)]]
**[[anesthesia, in vitro fertilization, and natural mating of Xenopus tropicalis (Conlon lab)]]
**[[dejellying embryos (Zorn lab)]]

*'''''Transgenics'''''
**[[transgenesis in Xenopus laevis - adapted from Amaya et al 2001 (Conlon lab)]]
**[[tadpole tail sampling for genomic DNA (Conlon lab)]]

*'''''In Situ Hybridization'''''
**[[in situ hybridization, brief version (Harland lab)]]
**[[whole-mount in situ hybridization (Conlon lab)]]
***[[in situ checklist (Conlon lab)]]
**[[fluorescent in-situs and FCIS (Vize lab)]]
***[[wholemount embryo FISH / Double FISH]]
***[[flourescin tyramide synthesis]]
***[[Cy3 tyramide synthesis]]
***[[FCIS]]
***[[chromosomal FISH-TSA]]

*'''''Immunohisochemistry'''''
**[[wholemount antibody staining (CSH Protocols) (Wallingford lab)]]
**[[wholemount antibody staining - alkaline phosphatase detection (Conlon lab)]]
**[[wholemount antibody staining - adapted from Dan Weeks' protocol (Conlon lab)]]
**[[immunostaining sectioned embryos (Conlon lab)]]
**[[hydrochloric acid antigen retrieval (Zorn lab)]]

*'''''Histology'''''
**[[cryosectioning Xenopus embryos (Conlon lab)]]

*'''''Other Embryo Staining Protocols'''''
**[[benzidine-peroxidase staining for blood (Conlon lab)]]
**[[benzidine-peroxidase staining for blood (Thomsen lab)]]
**[[apoptosis via TUNEL (Veenstra lab)]]
**[[TUNEL staining of whole Xenopus embryos - Harland protocol (Conlon lab)]]

*'''''Other Immuno Protocols and Protein Protocols'''''
**[[immunoprecipitation (Stukenberg lab)]]
**[[immunofluorescence with biotinylated antibodies (Stukenberg lab)]]
**Purifying Polyclonal Antibodies using Affinity Columns/Chromatography
***[[Making an Affinity Column]]
***[[Running an Affinity Column]]
**[[whole embryo protein extraction with freon (Conlon lab)]]
**[[SDS-PAGE gels (Conlon lab)]]
**[[western blot protocol (Conlon lab)]]
**[[silver staining protein gels (Stukenberg lab)]]

*'''''[[Oocyte Transfer Technique (Heasman/Wylie labs)]]'''''
**[[Part 1: Choosing Oligodeoxyribonucleotides (ODNs)]]
**[[Part 2: ODN Modification]]
**[[Part 3: Host Transfer Technique]]
**[[Part 4: Fertilization and Development]]

*'''''Xenopus Oocyte and Egg Extracts'''''
**[[crude interphase egg extract (Shechter lab)]]
**[[isolation of chromatin from egg extract and histone recovery (Shechter lab)]]
**[[preparation of cycling egg extract (Shechter lab)]]
**[[cycling extracts, mike's protocol (Stukenberg lab)]]
**[[cycling extracts, sara's protocol (Stukenberg lab)]]
**[[CSF extract, mike's protocol (Stukenberg lab)]]
**[[CSF extract, sara's protocol (Stukenberg lab)]]
**[[assaying H1 kinase in extracts (Stukenberg lab)]]

*'''''Xenopus Tissue Culture'''''
**[[tissue culture of Xenopus cell lines S3, XTC (Stukenberg lab)]]
**[[isolation of mitotic chromosomes from XTC cells (Stukenberg lab)]]
**[[in-vitro kinetochore-microtubule binding assays (Stukenberg lab)]]

3G8- pronephros and otic vesicle

2011-07-20T17:40:40Z

68.147.37.142:

'''Antigen:''' dissected pronephroi, specific target unknown

'''Xenbase Gene Page:'''

'''Epitope:'''

'''Host Species/Isotype:''' mouse monoclonal IgG

'''Antigen Species:''' Xenopus laevis

'''Applications:''' Immunofluorescence, immunohistochemistry

'''Antibody Name/Catalog Number:''' 3G8

'''Supplier:''' EXRC [http://www.port.ac.uk/research/exrc/antibodies/Antibodies/]

'''Comments:''' IP unknown, does not work on Westerns

[[File:3G8.jpg]]
Note- the blue stain in this image is by antibody 3G8, the red stain is by antibody 4A6 [http://www.xenbase.org/xenwiki/index.php/Monoclonal_4A6_-_pronephros]

'''Contributor:''' Generated and characterized by Elizabeth Jones [http://www.xenbase.org:80/community/person.do?method=display&personId=753]. References: Dev Bio 171:531- [http://www.xenbase.org/literature/article.do?method=display&articleId=19219]

'''Additional Information:''' '''Developmental Window:''' First detects target at stage 31, continues until latest stages tested. As this antibody stained only the lumen of the pronephric proximal tubules the shape of the organ looks odd in early stages and only begins to look like a pronephros at around stage 34 when a well luminized epithelial tubule network has formed.

'''Stains:''' proximal pronephric nephron, nephrostomes and otic vesicle,

'''Fixatives:''' formalin, MEMFA, TCA are all OK. Following fixation sample must be dehydrated with methanol for the antibody to work optimally. Others not tested. The target degrades over time in alcohol, so samples should be stored for less than one month post-fixation.

'''Sensitive to bleaching:''' No, though store time in methanol may be shortened by bleaching

'''Functions post in situ?:''' No

'''Optimized conditions:''' Very clean antibody, very stable, can be reused. Development in BCIP/NBT usually takes 30 minutes, in red, about 60 minutes. Works well in immunofluorescense. As the pronephros is only covered by a thin transparent epidermis it is not necessary to clear stained embryos. In cleared embryos both pronephroi are often in view, one in focus and one blurred, and this can make photography difficult see for example stage 32 sample above.

'''Other species:''' works on other anurans (Bombina), and also on axolotls when formalin plus 10% DMSO is used as the fixative, but it isn't nearly as clean as with Xenopus or Bombina. This is probably just a protocol problem. Reported to work on zebrafish.

'''XAO:'''

'''Reference(s):''' Dev Bio 171:531- [http://www.xenbase.org/literature/article.do?method=display&articleId=19219]

applications abbreviations:

WB - Western blotting,

IP - immunoprecipitation,

IF - immunofluorescence,

IHC - immunohistochemistry,

EMSA - electromobility shift assay,

ChIP - chromatin immunoprecipitation,

FCM - flow cytometry

3G8- pronephros and otic vesicle

2011-07-20T17:39:24Z

68.147.37.142:

'''Antigen:''' dissected pronephroi, specific target unknown

'''Xenbase Gene Page:'''

'''Epitope:'''

'''Host Species/Isotype:''' mouse monoclonal IgG

'''Antigen Species:''' Xenopus laevis

'''Applications:''' IF

'''Antibody Name/Catalog Number:''' 3G8

'''Supplier:''' EXRC [http://www.port.ac.uk/research/exrc/antibodies/Antibodies/]

'''Comments:''' IP unknown, does not work on Westerns

[[File:3G8.jpg]]
Note- the blue stain in this image is by antibody 3G8, the red stain is by antibody 4A6 [http://www.xenbase.org/xenwiki/index.php/Monoclonal_4A6_-_pronephros]

'''Contributor:''' Generated and characterized by Elizabeth Jones [http://www.xenbase.org:80/community/person.do?method=display&personId=753]. References: Dev Bio 171:531- [http://www.xenbase.org/literature/article.do?method=display&articleId=19219]

'''Additional Information:''' '''Developmental Window:''' First detects target at stage 31, continues until latest stages tested. As this antibody stained only the lumen of the pronephric proximal tubules the shape of the organ looks odd in early stages and only begins to look like a pronephros at around stage 34 when a well luminized epithelial tubule network has formed.

'''Stains:''' proximal pronephric nephron, nephrostomes and otic vesicle,

'''Fixatives:''' formalin, MEMFA, TCA are all OK. Following fixation sample must be dehydrated with methanol for the antibody to work optimally. Others not tested. The target degrades over time in alcohol, so samples should be stored for less than one month post-fixation.

'''Sensitive to bleaching:''' No, though store time in methanol may be shortened by bleaching

'''Functions post in situ?:''' No

'''Optimized conditions:''' Very clean antibody, very stable, can be reused. Development in BCIP/NBT usually takes 30 minutes, in red, about 60 minutes. Works well in immunofluorescense. As the pronephros is only covered by a thin transparent epidermis it is not necessary to clear stained embryos. In cleared embryos both pronephroi are often in view, one in focus and one blurred, and this can make photography difficult see for example stage 32 sample above.

'''Other species:''' works on other anurans (Bombina), and also on axolotls when formalin plus 10% DMSO is used as the fixative, but it isn't nearly as clean as with Xenopus or Bombina. This is probably just a protocol problem. Reported to work on zebrafish.

'''XAO:'''

'''Reference(s):''' Dev Bio 171:531- [http://www.xenbase.org/literature/article.do?method=display&articleId=19219]

applications abbreviations:

WB - Western blotting,

IP - immunoprecipitation,

IF - immunofluorescence,

IHC - immunohistochemistry,

EMSA - electromobility shift assay,

ChIP - chromatin immunoprecipitation,

FCM - flow cytometry